The role of Dot1L activity in chondrogenic differentiation

Dot1L 活性在软骨分化中的作用

基本信息

批准号：
10669804
负责人：
Rosaria M. Guzzo
金额：
$ 47.23万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-21 至 2027-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10669804
关键词：
Address Biological Assay Biology Bone Development Bone Growth Bone Morphogenetic Proteins CRISPR/Cas technology Cartilage Catalytic Domain Cell Differentiation process Cells ChIP-seq Chemicals Child Chondrocytes Chondrogenesis Chromatin Chromatin Remodeling Factor Complex Data Defect Development Disease Engineering Enzymes Epigenetic Process Epiphysial cartilage Female Functional disorder Future Gatekeeping Gene Expression Gene Expression Regulation Genes Genetic Transcription Goals Growth Growth and Development function Hindlimb Histones Hypertrophy Impairment In Vitro Knock-out Knowledge Limb Bud Limb structure Lysine Malignant Childhood Neoplasm Mesenchyme Methylation Methyltransferase Molecular Molecular Conformation Morphology Mus Mutant Strains Mice Outcome Study Pathologic Pathway interactions Pattern Phenotype Physiologic Ossification Physiological Play Post-Translational Protein Processing Process Proliferating Proteins Publishing Regulation Reporting Research Personnel Role Signal Pathway Signal Transduction Skeletal Development Technology Testing Therapeutic adverse outcome bone cartilage cell conditional knockout epigenetic regulation experimental study gene network genome wide methylation genome-wide analysis histone methyltransferase histone modification in vivo innovation insight long bone male methylation pattern mutant novel pharmacologic premature programs protein function single-cell RNA sequencing skeletal small molecule targeted treatment therapeutic target transcriptome sequencing transcriptomics

项目摘要

Abstract The goal of this new application is to define the role of the chromatin modifier Dot1L (Disruptor of telomeric silencing-1 like) in normal skeletal growth and development. Dot1L is the only enzyme that catalyzes the methylation of lysine 79 in histone 3 (H3K79), which plays an important role in the epigenetic regulation of gene expression. The only molecular function that has been demonstrated for Dot1L resides in its catalytic or methyltransferase (MT) domain, however new studies indicate that non-catalytic functions of Dot1L also contributes to the regulation of gene expression and cell differentiation. Currently, very little is known about how Dot1L regulates skeletal growth and development. This represents a critical gap in our knowledge because Dot1L targeted approaches are being studied as therapies for pediatric cancers. We recently reported that the conditional loss of Dot1L expression in limb mesenchyme induced an aberrant skeletal phenotype characterized by long bone shortening, and defects in growth plate (GP) chondrocyte proliferation. Interestingly, small molecule inhibition of Dot1L catalytic activity did not impair chondrocyte proliferation in vitro, suggesting an underlying but critical role for non-catalytic functions of Dot1L in these complex processes. Chemical inhibition of Dot1L in chondrogenic limb bud micromass assays resulted in premature chondrocyte hypertrophy through mis-regulation of the Bone morphogenetic protein (Bmp) signaling pathway. New in vivo data from our lab provides compelling evidence that a catalytic inactive Dot1L mutant protein can restore the cartilage GP dysfunction and long bone growth deficits in mice with conditional loss of Dot1L function in limb mesenchyme. Together, these data, support our novel central hypothesis that Dot1L regulates long bone growth at the GP through: i) non-catalytic activities that support chondrocyte proliferation; and ii) MT-dependent activities which restrict chondrocyte maturation. We have assembled a strong team of investigators with expertise in skeletal biology, epigenetics, and Dot1L biology to address this novel hypothesis. In Aim 1, we will establish the functional requirement for Dot1L catalytic activity in endochondral bone growth in vivo, using novel Dot1L MT mutant mice. Our studies will determine whether a catalytic-dead Dot1L mutant can rescue skeletal defects in Dot1L cKOPrrx1 mice. We will apply single cell transcriptomic analyses to identify novel Dot1L-regulated genes and pathways. In Aim 2, we will define the regulatory functions of Dot1L that provide stage-specific control of chondrogenic differentiation. Using Dot1L knockout versus MT mutant cells, mechanistic studies will assess the direct contribution of Dot1L catalytic versus non-catalytic functions to chondrocyte proliferation versus hypertrophy. Lastly, ChIPseq experiments will identify genome-wide methylation patterns (H3K79me2) associated with chondrocyte proliferation and maturation. Outcomes from these studies are expected to generate new knowledge on Dot1L during normal skeletal growth and development, with implications for targeting Dot1L therapeutically.

抽象的这个新应用的目标是定义染色质修饰剂 Dot1L（端粒破坏者）的作用沉默-1样）在正常骨骼生长和发育中。 Dot1L 是唯一能够催化组蛋白 3 (H3K79) 中赖氨酸 79 的甲基化，在基因的表观遗传调控中发挥重要作用表达。 Dot1L 已被证明的唯一分子功能在于其催化或甲基转移酶 (MT) 结构域，然而新的研究表明 Dot1L 的非催化功能也有助于基因表达和细胞分化的调节。目前，人们对如何 Dot1L 调节骨骼生长和发育。这代表了我们知识中的一个关键差距，因为 Dot1L 靶向方法正在研究作为儿科癌症的治疗方法。我们最近报道称肢体间充质中 Dot1L 表达的条件性丧失诱导了异常的骨骼表型长骨缩短和生长板（GP）软骨细胞增殖缺陷。有趣的是，小分子抑制 Dot1L 催化活性不会损害体外软骨细胞增殖，这表明潜在的但 Dot1L 在这些复杂过程中的非催化功能发挥着关键作用。 Dot1L 的化学抑制软骨形成肢芽微量测定通过错误调节导致软骨细胞过早肥大骨形态发生蛋白 (Bmp) 信号通路的研究。我们实验室的新体内数据提供了令人信服的有证据表明催化失活的 Dot1L 突变蛋白可以恢复软骨 GP 功能障碍和长骨肢体间充质 Dot1L 功能条件性丧失的小鼠的生长缺陷。这些数据共同支持我们新的中心假设是，Dot1L 通过以下方式调节 GP 的长骨生长：i) 非催化活性支持软骨细胞增殖； ii) 限制软骨细胞成熟的 MT 依赖性活动。我们组建了一支强大的研究团队，拥有骨骼生物学、表观遗传学和 Dot1L 生物学方面的专业知识来解决这个新颖的假设。在目标 1 中，我们将建立 Dot1L 催化活性的功能要求使用新型 Dot1L MT 突变小鼠进行体内软骨内骨生长。我们的研究将确定是否催化死亡 Dot1L 突变体可以挽救 Dot1L cKOPrrx1 小鼠的骨骼缺陷。我们将应用单细胞转录组分析以确定新的 Dot1L 调控基因和通路。在目标 2 中，我们将定义 Dot1L 的调节功能提供软骨形成分化的阶段特异性控制。使用 Dot1L 敲除细胞与 MT 突变细胞相比，机制研究将评估 Dot1L 催化与 MT 突变细胞的直接贡献对软骨细胞增殖与肥大的非催化功能。最后，ChIPseq 实验将确定与软骨细胞增殖和成熟相关的全基因组甲基化模式（H3K79me2）。这些研究的结果预计将产生关于正常骨骼生长期间 Dot1L 的新知识和发展，对靶向 Dot1L 治疗具有影响。